Ite for cancer intervention and eradication

ABSTRACT

A method of cancer intervention or eradication by administering an effective amount of an endogenous ligand for the aryl hydrocarbon (Ah) receptor (AhR) named ITE or one of its analogs (the active ingredient) to a subject with cancer is disclosed. An effective dose and dosing frequency of the active ingredient are determined by measuring its blood levels of the subject after dosing. The active ingredient formulated with a carrier system is applied topically, enterally, or parenterally to the subject. The formulated drug can also be administered together with one or more of other cancer therapeutic agents. A maintenance dosing is provided after the subject is free of cancer to insure the cancer eradication. Subjects with cancers of prostate, liver, lung, ovarian, and breast are preferably accepted for treatment.

TECHNICAL FIELD

Cancer Therapy; Cancer Treatment; Cancer Intervention; CancerEradication; Cancer Biology; Oncology; Therapeutics; Pharmaceuticals;Biopharmaceuticals.

BACKGROUND ART

The aryl hydrocarbon (Ah) receptor (AhR) is a ligand inducibletranscription factor, a member of a so-called basichelix-loop-helix/Per-Arnt-Sim (bHLH/PAS) superfamily. Upon binding toits ligand, AhR mediates or interacts with a series of biologicalprocesses as well as some adverse effects including cell division,apoptosis (programmed cell death), cell differentiation, actions ofestrogen and androgen, adipose differentiation, hypothalamus actions,angiogenesis, immune system stimulation or suppression, teratogenicity,tumorigenicity, tumor initiation, tumor promotion, tumor progression,chloracne, wasting syndrome, and actions of other hormonal systemsbeside the expression of genes of P450 family andothers^([1,2,3,4,5,6,7,8].) The liganded receptor participates inbiological processes through translocation from cytoplasm into nucleus,heterodimerization with another factor named Ah receptor nucleartranslocator, attachment of the heterodimer to the regulatory regiontermed Ah response element of genes under AhR regulation, and theneither enhancement or inhibition of transcription of those genes.

The AhR happens to be able to bind, with different affinities, toseveral groups of exogenous chemicals (thus artificial ligands) such aspolycyclic aromatic hydrocarbons exemplified by 3-methylchoranthrene(3-MC) and halogenated aromatic hydrocarbons typified by2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The receptor system has beenstudied so far with its artificial ligands. While studies with those AhRartificial ligands helped in advancing our understanding toward thereceptor system, thorough elucidation of the physiological roles thesystem plays and the potential therapeutic benefits the system may offerare impossible without identification of the AhR physiological ligand.As the first step toward this goal, an endogenous ligand for thereceptor has been identified. The endogenous ligand, or physiologicalligand, or natural hormone, for the AhR was identified as2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE)^([9,10]).

Even though most of the artificial ligands for AhR are environmentaltoxins^([1,2,3]) and thus cannot be used as therapeutic agents, for thepurpose of understanding functions of liganded AhR, its artificialligands such as TCDD, 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF),8-methyl-1,3,6-trichlorodibenzofuran (8-MCDF), and those derived fromindole or tryptophan were used to reveal that the liganded AhR was ableto inhibit the metastasis of prostate tumors in a strain of transgenicmice^([11]) and the growth of carcinogen induced rat mammarytumors^([12,13,14]), human breast tumor cell xenografts^([15,16]), andtumors caused by gene mutations^([17]).

As a natural ligand for AhR, ITE is an excellent agent in targetingprecisely and specifically the receptor. The consequence of thetargeting, however, is unpredictable from what we have learned so farfrom the behaviors of those artificial ligands for AhR, with someresults showing anitcancer potentials^([12,13,14,15,16]) while otherstumor initiation, promotion, and progression^([8,18,19,20,21]). From thefact that it is antiangiogenic, ITE might be useful in cancertherapy^([7]). The property of antiangiogenesis alone, however, will notautomatically qualify ITE as an effective anticancer agent. There arecountless examples proving the point^([22,23,24,25]). Manyantiangiogenic agents failed to perform as therapeutic agents^([22]) andlots of others even accelerated tumor invasion and metastasis^([26,27])due probably to the stress produced by the agents: limited supply ofoxygen and nutrients to the tumors. The antiangiogenic therapy isactually a concern since it may even reduce overall survival, the goldenstandard for cancer therapy, due to possibly an acceleratedmetastasis^([23]). From the anticancer property of liganded AhR revealedby its artificial ligands^([12,13,14,15,16]), there is no guarantee thatITE, once bound to the same receptor, may also be able to do evenpartially what these artificial ligands could do without mentioning thefact that most of those artificial ligands or their metabolites may behighly toxic to those cancer cells being tested. In that sense, thoseartificial ligands or their metabolites may merely serve asnon-discriminative cytotoxic agents, killing cancer cells, not even theresults of targeting the Ah receptor.

In addition, a critical factor determines what a liganded receptor willdo is the final three dimensional (3D) structure the liganded receptorassumes since it is the 3D structure that dictates how many differentcellular factors the liganded receptor will interact with and how theseinteractions should be carried out to conduct the processes of life. Thefinal 3D structure the liganded receptor assumes is, in turn, solelyshaped by the 3D structure of the ligand for the receptor in a givenbiological system. That is the fundamental basis explaining why the 3Dstructure of a ligand is so crucial in directing its receptor mediatedbiological and pharmacological processes. Furthermore, ligands withdifferent structures metabolize differently and their differentmetabolites will certainly interfere with the biological processesdifferentially. It is obvious, therefore, that ligands with different 3Dstructures could then certainly lead to completely different biologicalconsequences even if they can bind to the same receptor.

The validity of the point can be easily established through theoreticreasoning above and of course through illustration of literature dataalso. For example, even though both TCDD and6-formylindolo[3,2-b]carbazole (FICZ) are high affinity ligands for AhR,TCDD is found to stimulate Treg cell differentiation, thus suppressingthe immune system, while FICZ promote Th17 cell differentiation, thusstimulating the immune system^([28]). In another example, both TCDD andITE are high affinity ligands for AhR but while TCDD induced cleftpalate, hydronephrosis, and thymic atrophy, ITE did not do any ofthese^([29]). More examples can be easily found in theliterature^([30,31,32,33,34]). It is, therefore, not obvious at all thatITE will be a good anticancer agent from those studies with artificialligands for AhR to show their anticancer property^([12,13,14,15,16]) oreven the study with ITE to prove its antiangiogenic property^([7])without an extensive research program with different experimental modelsand systems to find out a clear answer.

The situation prompted us to investigate whether ITE or one of itsstructural analogs could be used efficaciously and safely in treating oreradicating cancer. The present invention fully discloses a method ofusing the newly discovered endogenous Ah receptor ligand ITE or one ofits structural analogs as a therapeutic agent in cancer intervention oreradication.

SUMMARY OF INVENTION Technical Problem

There are two serious problems with current cancer therapies in themarket. The first is severe side effects and toxicity. The second isvery limited efficacy. Consequently, cancer is still the second leadingcause of death in the United States and areas of the world.

The majority of current therapeutic agents for cancer, in both cytotoxicand noncytotoxic categories, are chemicals foreign to the human body. Asa result, the body tries extremely hard to get rid of them usingwhatever metabolic ways available. Since our body does not have anatural and safe way of metabolizing those foreign chemicals, somenonspecific oxidation reactions then are used as major means ofmetabolism. The consequence is that the elimination processesunavoidably generate a lot of chemically active intermediates orradicals, which will assault also normal cellular substances including,but not limited to, that of immune system's in the body, leading toserious side effects, toxicity, and weakened immune system. Since mostof these agents were designed by humans, not nature, they have very highchances to bind to and interact with other cellular factors (including,but not limited to, receptors, enzymes, other proteins) than theirexpected targets in the body. These “off-target” bindings andinteractions account for significant opportunities for side effects.

The effectiveness of cytotoxic agents for cancer therapy is mainlylimited by their indiscriminate toxicity to normal cells and tissuesincluding, but not limited to, that of immune system's. The weakenedimmune system, as expected, makes it impossible to launch an organizedassault on cancer cells. The efficacy of noncytotoxic agents, whichtarget specific functions important for the survival of cancer cells, islimited by their single mechanism based strategy. An important hallmarkof cancer, however, is their constant genetic changes or mutations. Oncea cancer cell changes into a state that it is no longer dependent on aspecific function a therapeutic agent targets for survival, the efficacyof the agent will then be lost immediately.

Solution to Problem

The situation thus calls for the emergence of a novel therapeutic agentthat can assault cancers with multiple combating capabilities forsustained potency, help immune system at the same time to organize anorchestrated attack on cancers and clean up individual cancer cells fora possible cancer eradication, and limit the chance of “off-target”interaction and metabolize itself safely for low side effect(s). Thenewly discovered Ah receptor endogenous ligand ITE or one of itsstructural analogs is capable of satisfying the stringent requirementsset forth.

Advantageous Effects of Invention

The most important advantage of the present invention is ITE's or one ofits structural analogs' multiple cancer assaulting capabilities to defythe consequence of cancer cells' constant genetic changes. ITE has beendemonstrated to inhibit angiogenesis^([7]). In literature, the Ahreceptor (AhR) liganded with its artificial ligands (not ITE) was shown(possible negative effects of those artificial ligands are ignored herefor the moment) to be able to inhibit cell division^([35,36,37]),promote programmed cell death (apoptosis)^([38,39,40]), induce celldifferentiation^([41,42,43]), and block actions of estrogen^([6,44]) andandrogen^([45,46]). Recently, AhR liganded with artificial ligands (notITE) has been demonstrated to be able to induce the differentiation ofimmune T cells^([28,47]), useful for the immune system in organizingassault on pathogens and cancers. If ITE, or one of its structuralanalogs, when bound to AhR, can also have one or more of the functionsmentioned plus its antiangiogenic property, the multiple cancerassaulting capabilities may make its cancer therapeutic potencysustainable. The sustainability of the potency of ITE or one of itsanalogs plus its potential ability of stimulating the immune systemwould not only enhance dramatically the efficacy of the cancer therapybut also make cancer eradication a possibility. The data presented inDrawings and Examples clearly verify the theoretical analysis above.

A huge benefit of using ITE over others in the market is its possibilityof low side effect(s) beside its sustainable efficacy backed by itsmultiple cancer assaulting capabilities. Contrary to those chemicals,including those AhR artificial ligands and the agents used in currentcancer therapies, foreign to the human body and designed by humans, ITEis a natural hormone designed by nature and so nature may have designedand implemented a natural and safe way for its metabolism. Its metabolicprocess thus will cause less or even no problem to the body. This meansthat it may be low in side effect(s) caused by its metabolism. Anotherimportant reason for possible low side effect is that the binding of thenatural hormone to its receptor (AhR) is very specific and precise sinceit is designed by nature, not humans. The natural hormone ITE, otherthan those human designed chemicals, will then have a low chance ofbinding to and interacting with other cellular factors to provoke“off-target” problems, important opportunities for side effects. Theexperimental data described in Drawings and Examples support the point.

Another important issue in cancer therapy is that it is highly desirablefor a therapeutic agent specifically working in cancer cells instead ofnormal cells to enhance its potency and reduce side effects. This typeof specificity can be achieved if there are more target molecules theagent binds in cancer cells than in normal cells. The target moleculefor ITE and its analogs is AhR. In the literature, the AhR was reportedto be highly concentrated in pancreatic cancer tissues from patients butvery diluted in all normal pancreatic tissues examined^([48]).Similarly, the concentrated AhR is also documented with prostatecancer^([49,50]) and gastric cancer^([51]). This means that thetherapeutic specificity of ITE and its structural analogs could beachieved in these reported types of cancers at least.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D show the growth inhibition of human cancer cell line LNCapxenografts in response to doses, routes, and schedules of ITEadministration, wherein FIG. 1A shows the degrees of growth inhibitionof LNCaP xenografts (mean+SEM, n=8) in response to ITE doses of 0(vehicle, DMSO), 0.1, 1, 10, 20, and 40 mg/kg b.w. (i.p. injection,every 12 hours for 28 continuous days), and wherein FIG. 1B shows thelow toxicity response of the xenograft-bearing mice to the treatmentjudged by their body weight changes (mean+SEM, n=8), and wherein FIG. 1Cshows PK (Pharmacokinetic) profiles of ITE administered (single dosing)i.v., i.p., and p.o. with vehicles used, dosing levels, and AUC's (Areaunder Curves) as indicated, and wherein FIG. 1D shows the inhibition ofLNCaP xenograft growth by ITE at different doses (40 or 80 mg/kg b.w.),schedules (once or twice daily), and routes (i.p. or p.o.) ofadministration as specified.

FIGS. 2A-2D show shows ITE (diamond) or ITK (one of ITE structuralanalogs, square) efficacy in inhibiting the growth of xenografts ofhuman prostate (LNCaP), liver (HepG2), ovarian (OVCAR-3), and breast(MCF-7) cancer cell lines (i.p. once daily), wherein FIG. 2A showsinhibition of LNCaP xenograft growth by ITE or ITK at 20 mg/kg for both,and wherein FIG. 2B shows growth inhibition of HepG2 xenografts by ITEor ITK at 80 mg/kg for both, and wherein FIG. 2C shows ITE or ITKinhibiting OVCAR-3 xenograft growth at a dose of 80 mg/kg for both, andwherein FIG. 2D shows a growth inhibition of MCF-7 xenografts by ITE ata dose of 20 mg/kg.

FIGS. 3A-3D show cancer inhibition and eradication by ITE (i.p. oncedaily) in a syngeneic murine Lewis lung cancer (LLC) model, wherein FIG.3A shows aggressive growth of LLC tumors and ITE inhibition of tumorgrowth at a dose of 20 mg/kg, and wherein FIG. 3B shows a betterinhibition of LLC tumor growth by ITE at 80 mg/kg (i.p. once daily) sothat a treatment program of 28 days plus one more week of post injectionobservation could be finished, and wherein FIG. 3C shows one mouse (No.33, diamond) from the ITE group (square) initiating its tumor shrinkageupon the start of the treatment phase, becoming tumor-free at day 13 inthe treatment, keeping the tumor-free status during the rest of thetreatment phase, and being still tumor-free in an entire one month ofthe observation phase, and wherein FIG. 3D shows body weight changes ofmouse No. 33 (diamond) together with that of ITE (square) and vehiclecontrol (circle) groups.

DESCRIPTION OF EMBODIMENTS

All technical and scientific terms used herein are the same as thosecommonly used by one of ordinary skill in the art to which the presentinvention pertains unless defined specifically otherwise. It isunderstood that other materials and methods similar or equivalent tothose described herein can also be used in the practice or in thetesting of the present invention but only preferred materials andmethods are described below.

The present invention is a method of cancer intervention or eradicationwith an endogenous aryl hydrocarbon (Ah) receptor (AhR) ligand ITE orone of its structural analogs. ITE or one of its analogs (the activeingredient) can be formulated with one or more pharmaceuticallyacceptable carrier(s) (the carrier system). The carrier system isconsisted of inert materials useful for administering the activeingredient, preferably sterile and nontoxic. The carrier system shouldbe compatible with the active ingredient and can be in a form of solid,liquid, or gas. The properly formulated active ingredient can then beadministered topically, enterally, or parenterally to a subject withcancer. It can be provided, for example, in a form of cream, capsules,tablets, lozenges, or injectables. Other compatible ingredients such aspreservatives, if needed, could be co-formulated with the activeingredient.

In a preferred intervention program, subjects with cancers of prostate,liver, lung, ovarian, and breast are preferably accepted for treatmentwith ITE or one of its structural analogs. This is by no mean to limitthe therapeutic scope, however. Given the multiple cancer assaultingcapabilities that ITE and one of its analogs possesses plus thepossibility of stimulation of a subject's immune system to attackcancers and clean up individual cancer cells for possible cancereradication, the therapeutic scope is envisioned to be expanded quicklyin future trials.

In the preferred intervention program, the effective dose range of ITEor one of its structural analogs is determined by measuring thesubject's blood concentration of ITE or one of its structural analogsunder a specified dosing regimen to establish a concentration-timeprofile, consulting with an established correlation between the similarconcentration-time profiles and effects on cancer inhibition oreradication, which built during a trail or trials as that illustrated inExamples, and balancing the therapeutic effects achievable with thepossible toxicity to the subject and health condition or physicaldurability of the subject. The dosing frequency of ITE or one of itsstructural analogs is decided similarly as described for thedetermination of a dose range above. Currently, once a dayadministration either enterally or parenterally is proposed aspreferable with ITE. The dosing will be continued until the subject isfree from the cancer. It is preferable to provide a maintenance dosing,whose duration is directed by a trial or trials, after the subject isfree of cancer to insure its complete elimination or eradication.

In another preferred intervention program, ITE or one of its structuralanalogs may be administered in combination with one or more of othercancer therapeutic agents, preferably aiming different therapeutictargets other than AhR. ITE or one of its structural analogs can beformulated either independently from or together with one or more of theother said agents. ITE or one of its structural analogs can beadministered either at the same schedule with or different from that ofone or more of the other said agents. The proportioning of ITE or one ofits structural analogs to one or more of the other cancer therapeuticagents will be directed by a well-designed trial or trials. Combiningthe therapy of ITE or one of its analogs with one or more of the othercancer therapeutic agents, may further enhance the efficacy. There arelots of examples to show the benefits of combination therapy.

In those preferred intervention programs, the active ingredient is thearyl hydrocarbon (Ah) receptor (AhR) endogenous ligand ITE with thefollowing structural formula (Structural Formula 1):

In those preferred intervention programs, the active ingredient can beselected from two especially useful structural analogs of ITE. The saidtwo analogs are envisioned to increase their stability and then extendtheir half-life in the subjects' systems since either a ketone or thiolester functional group replaces the normal (oxygen) ester, targetedeasily by numerous esterases in biological systems, in the structure ofITE. The extended half-life may translate into higher efficacy and/orlonger duration of potency in cancer intervention. The ketone analog(thus termed ITK) of ITE is of the following structural formula(Structural Formula 2):

Whereas the structural formula of the thiol (S, sulfur) ester analog(thus termed ITSE) of ITE is as follows (Structural Formula 3):

In those preferred intervention programs, the active ingredient can befurther selected from the other structural analogs of ITE, specified bythe following structural formula (Structural Formula 4):

wherein:

X and Y, independently, can be either O (oxygen) or S (sulfur);

R_(N) can be selected from hydrogen, halo, cyano, formyl, alkyl,haloalkyl, alkenyl, alkynyl, alkanoyl, haloalkanoyl, or a nitrogenprotective group;

R₁, R₂, R₃, R₄, and R₅ can be independently selected from hydrogen,halo, hydroxy (—OH), thiol (—SH), cyano (—CN), formyl (—CHO), alkyl,haloalkyl, alkenyl, alkynyl, amino, nitro (—NO₂), alkoxy, haloalkoxy,thioalkoxy, alkanoyl, haloalkanoyl, or carbonyloxy;

R₆ and R₇, can be independently selected from hydrogen, halo, hydroxy,thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl, amino, nitro,alkoxy, haloalkoxy, or thioalkoxy; or

R₆ and R₇, independently, can be:

wherein R₈ can be selected from hydrogen, halo, cyano, alkyl, haloalkyl,alkenyl, or alkynyl; or

R₆ and R₇, independently, can be:

wherein R₉ can be selected from hydrogen, halo, alkyl, haloalkyl,alkenyl, or alkynyl; or

R₆ and R₇, independently, can be:

wherein R₁₀ can be selected from hydrogen, halo, hydroxy, thiol, cyano,alkyl, haloalkyl, alkenyl, alkynyl, amino, nitro; or

R₆ and R₇, independently, can also be:

wherein R₁₁ can be selected from hydrogen, halo, alkyl, haloalkyl,alkenyl, or alkynyl.

EXAMPLES

Examples from preclinical animal studies will further help theembodiment of the present invention. Use of ITE in inhibition of humanprostate cancer growth (Example 1), use of ITE or ITK (one of ITEanalogs) in inhibiting the growth of more human cancer types (Example2), use of ITE in possible cancer eradication (Example 3), and ITEtoxicity monitoring (Example 4) will be demonstrated.

Example 1 Materials

Male BALB/c nude mice (Mus musculus), 6 to 8 weeks of age, wereindividually marked by ear coding. The animals were kept in laminar flowrooms at a constant temperature of 20 to 26° C. and humidity of 40 to70% with 1 animal in each polycarbonate cage (300 mm×180 mm×150 mm). Thebedding material was corn cob, which was changed twice weekly. Animalshad free access to sterile dry granule food and sterile drinking waterduring the entire study.

ITE was synthesized by KNC Laboratories Co., Ltd. (Tokyo, Japan). Thelot number of the compound is 086-009-2-1 (as lot No.: AHR-001 for AhRPharmaceuticals). The DMSO (Cat. No.: 0231-500 ML) was manufactured byAMRESCO (Solon, Ohio, USA). The Labrasol was purchased from Gattefosse(Saint-Priest, France) and the PEG 400 was supplied by Sigma (St.Louise, Mo., USA).

Methods Efficacy Studies

All the procedures related to animal handling, care, and the treatmentin the study were performed following guidelines approved by anInstitutional Animal Care and Use Committee (IACUC) of Crown Bioscience,Inc. (Santa Clara, Calif., USA, a contract research organization wehired) based on the guidance of the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC). Animals were checkedfor any effects of tumor growth and drug treatment on normal behaviorsuch as mobility, food and water consumption, body weight gain/loss(gross body weights were measured twice weekly), eye/hair matting, andany other abnormal effect. Death and observed clinical signs wererecorded on the basis of the number of animals within each group.Individual animals with a tumor volume exceeding 3000 mm³ or animals ofa group with a mean tumor volume exceeding 2,000 mm³ were euthanized. Inaddition, animals showing signs of severe distress and/or pain, droppingbody weight more than 20% from that at the start of treatment, or losingthe capability of accessing adequate food or water were humanelysacrificed.

The human prostate cancer cell line LNCaP (ATCC, American Type CultureCollection, Manassas, Va., USA) were maintained in vitro as monolayerculture in RPMI-1640 medium supplemented with 10% fetal bovine serum(FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamineat 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells wereroutinely subcultured twice a week. The cells growing in an exponentialphase were harvested and counted for tumor inoculation.

Each mouse was inoculated subcutaneously at the right flank with theLNCaP cells (1×10⁷) in 0.1 ml of PBS for tumor development. When a meantumor volume reached around 150 mm³, the tumor-bearing mice were dividedinto homogeneous blocks based on their tumor volumes followed by arandomization of mice in each block into treatment groups (thusminimizing variations in tumor response to treatments due to thedifferential in initial mean tumor volumes). Each treatment group wasconsisted of 8 tumor-bearing mice. Vehicle (DMSO) or ITE in the vehicleat specified doses were administered to the mice by either i.p.(intraperitoneal) or p.o. (oral) injection once or twice daily for 28continuous days as indicated.

Tumor volume was measured twice weekly in two dimensions using a caliperand the volume was calculated with a formula of: V=0.5 a ×b², where aand b are the long and short diameter (in mm) of a tumor, respectively.The tumor volume was then used for calculations of both TGI (TumorGrowth Inhibition) and TGD (Tumor Growth Delay). The TGI was determinedby: TGI=ΔT/ΔC×100%, where AT was a difference between the mean tumorvolume at a specified day of observation and that at the day treatmentstarts (day 1) for a drug treated group whereas ΔC was the samedifference measured for the control group. The TGD was calculated as:TGD=T−C, where T was the time (in days) required for tumors in a drugtreated group to reach a predetermined mean tumor volume and C the time(in days) in the control group to reach the same volume. A tumor weightwas derived by equating 1,000 mm³ in volume to 1,000 mg in weight. A netbody weight was then derived by subtracting a tumor weight from acorresponding gross body weight with the tumor.

Summary statistics, including mean and the standard error of the mean(SEM), are provided for the tumor volume of each group at each timepoint. Statistical analysis of difference in tumor volume among groupswas conducted on a data set either at the best therapeutic time point orat the final dosing day as indicated. The tumor volume data werelog-transformed and evaluated using a one-way ANOVA followed by Tukey'stest when significance was observed. All data were analyzed using SPSS16.0 and p<0.05 was considered to be statistically significant.

PK Studies

Male nude mice were also used in PK (pharmacokinetic) studies. For i.v.injection, ITE at 1 mg/kg b.w. was administered with DMSO as vehicle viatail vein. For i.p. Injection, ITE at 10, 40, and 80 mg/kg b.w. wasdelivered with DMSO via lower left abdominal quadrant. In p.o.injection, ITE at 40 and 80 mg/kg b.w. were administered with a vehicleof Labrasol:PEG 400 (2:8, v/v) via oral gavage. Every 15 mice were givena single injection at each dosing level and every 3 of the dosed micewere used to collect blood sample at each time point (0, 0.083, 0.25,0.5, 1, 2, 4, 8, 24 hr.). Animals were rotated to be sampled twice eachbut the duration between the two sampling times was at least 110 min.The animal was anesthetized under Isoflurane and restrained manually.Approximately 150 μl of whole blood at each time point is collected (viaretro-orbital puncture) into a K₂-EDTA tube. Blood samples were put onice and processed to plasma (4,000 g, 5 min, 4° C.) within 15 min postsampling. Plasma samples were stored at −80° C. until analysis. Analiquot of 20 μl plasma sample was added with 20 μl of an internalstandard (Glipizide, 500 ng/ml in ACN, for extraction efficiency) to 120μl of ACN (acetonitrile). The mixture was vortexed at 1,500 rpm for 2min. and then centrifuged at 12,000 rpm for 5 min. Five (5) μl of thesupernatant was injected into an LC-MS/MS system (API 4000, Foster City,Calif., USA). A Gemini-C18 column (2.0×50 mm, 5 μm) was used and the LC(liquid chromatography) was run at a flow rate of 0.45 ml/min. with thefollowing program:

Time (min) 0.0 0.2 1.8 2.8 2.9 4.0 Pump A (%) 95 95 2 2 95 Stop Pump B(%) 5.0 5.0 98 98 5.0 Stopwhere Pump A was for 1 mM NH₄OAc (ammonium acetate) in water plus 0.025%FA (formic acid) whereas Pump B for 1 mM NH₄OAC in acetonitrile plus0.025% FA. The negative ionization process of the mass spectrometry wasoperated at an APCI (Atmospheric Pressure Chemical Ionization) modewhile the detection at a MRM (Multiple Reaction Monitoring) mode. ITEwas identified by recognizing an LC retention time of 2.5 min. and twomass peaks at 285.0 (before collision) and 142.0 m/z (after collision)while the internal standard of 2.35 min. and two mass peaks at 444.3(before collision) and 319.3 m/z (after collision). ITE was quantifiedby a standard curve generated every time by a series of known quantitiesof ITE running through both the extraction/precipitation process aftermixing with mouse plasma and the LC-MS/MS system. The WinNonlin V5.2statistics software (Pharsight Corporation, California, USA) was used togenerate PK parameters such as C_(max), T_(max), T_(1/2), and AUC (Areaunder Curve) etc. using a non-compartmental model.

Results and Discussion

Treatment with ITE at doses of 1,10, 20, and 40 mg/kg b.w. (i.p., every12 hr. for 28 continuous days, DMSO as vehicle, 0.5 ml/kg b.w. asinjection volume) produced significant anticancer activities with aclear dose-effect relationship (FIG. 1A). The TGI's (Tumor GrowthInhibition) were calculated as 52%, 31%, 26%, and 22% at day 28 (n=8;p<0.048, 0.007, 0.004, and 0.004), respectively, for the dosing series.The TGD (Tumor Growth Delays) of 3, 10, 12, and 16 days, respectively,at a tumor size of 600 mm³ were attained by the series. ITE at 0.1 mg/kgb.w. didn't produce a statistically significant anticancer activity(n=8, TGI=74% at day 28, p<0.623). Judging from the body weight changesof the tumor-bearing mice, ITE treatment did not seem to provokesignificant toxic response (FIG. 1B).

To understand pharmacokinetic (PK) behavior of ITE and direct furtherefficacy studies, ITE was administered to nude mice in different routesand at different levels. ITE PK profiles are depicted in FIG. 1C. ITE inDMSO at 1 mg/kg b.w. delivered by a bolus i.v. injection was degradedvery quickly with an estimated half-life of 6 min. An estimated AUC forthe route was 256 hr·ng/ml. ITE in DMSO administered by i.p. injectionimproved its half-life while the efficiency of absorption was below 10%compared with that of i.v. injection. For example, ITE half-life for 10,40, and 80 mg/kg b.w. of i.p. injection were 1.13, 1.61, and 5.17 hr.,respectively, while AUC for the series was 197, 332, 499 hr·ng/ml,respectively. ITE in Labrasol:PEG 400 (2:8, v/v) delivered via p.o.route had even lower absorption efficiency (around 1%) while kept thehalf-life to the levels that achieved by i.p. injection. The AUC for thedosing levels of 40 and 80 mg/kg b.w. of p.o. injection was 107 and 97hr·ng/ml, respectively (FIG. 1C).

Based on the results from PK studies, the schedule, dosing level, androutes of ITE administration were further explored. When a total dailydose was kept the same, the dosing schedule of either once or twicedaily for the i.p. route resulted in comparable efficacy in inhibitionof cancer growth (e.g. 80 once vs. 40 mg/kg b.w. twice daily, FIG. 1D).Further raising ITE dose to 80 mg/kg (twice daily, i.p.) seemed tofurther improve the TGI from that of 40 mg/kg (twice daily, i.p.). TGIfor 80 mg/kg (twice daily, i.p.) were the best so far obtained, 12% atday 28 and 16% at the last day, for example. Even though absorptionefficiency of ITE via p.o. route at a dose of 80 mg/kg was much lowerthan that of 10 mg/kg i.p. in terms of AUC (FIG. 1C), 80 mg/kg p.o.daily was similar to that of 40 mg/kg i.p. daily in terms of cancergrowth inhibition (TGI=46% at day 24, for example) during the firstthree weeks or so. From the PK studies, ITE plasma level of 80 mg/kgp.o. was lower than that of 10 mg/kg i.p. at the initial hours butbecame higher than that of 10 mg/kg i.p. and even 40 mg/kg i.p. duringhour 3 to 8 post injection (FIG. 1C). That may be the reason behind theresults of p.o. injection during the first three weeks. For the p.o.injection, its therapeutic efficacy somehow would not hold longer thanthat of i.p. injections toward and post the end of treatment (FIG. 1D).

Example 2 Materials and Methods

The culture and inoculation of human prostate cancer cell line LNCaPwere as described in Example 1. The manipulation for human liver cancercell line HepG2 (ATCC) was similar as that for LNCaP except that DMEM(instead of RPMI-1640) medium was used, the L-glutamine was not used,and 2×10⁶ cells were used for inoculation into female nude mice. Thehandling of human ovarian cancer cell line OVCAR-3 (ATCC) was the sameas that of LNCaP except that the DMEM medium was used and 5×10⁶ cellswere used for inoculation into female nude mice. The human breast cancercell line MCF-7 (CL-161) is a cloned line from MCF-7 (ATCC) and thegrowth of its xenografts no longer needs exogenous supply of estrogen.The culture of the MCF-7 cells was similar as that of LNCaP except thatMEM medium supplemented with 1 mM non-essential amino acids, 1 mM sodiumpyruvate, and 0.01 mg/ml bovine insulin was used to replace RPMI-1640medium. The inoculation of MCF-7 cells was the same as that of LNCaPexcept that 0.1 ml of PBS with Matrigel (1:1) and female mice were usedfor tumor development.

Source of ITE is the same as that described in Example 1. The compoundITK (Structural Formula 2), one of ITE structural analogs, wassynthesized by Shanghai ChemPartner Co., Ltd. (Shanghai, China). The lotnumber was: AhR-ITK-001.

Results and Discussion

ITK (one of ITE structural analogs) was shown to be efficacious at 20mg/kg b.w. (i.p. once daily) and performed even better than ITE in thesame regimen in human prostate cancer (LNCaP) xenograft model (FIG. 2A).The TGFs (Tumor Growth Inhibition) were 51% (p<0.003, n=8) and 64%(p<0.021, n=8) for ITK and ITE, respectively, at day 28. The TGD's(Tumor Growth Delay) for ITK and ITE were 16 and 8 days, respectively,at a tumor volume of 1,000 mm³.

Both ITE and ITK at 80 mg/kg (i.p. once daily) demonstrated goodefficacy in inhibiting the growth of human liver cancer (HepG2)xenografts. The performance of ITE and ITK was very comparable in thismodel (FIG. 2B). The TGFs for ITE and ITK were 25% (p<0.001, n=8) and22% (p<0.001, n=8), respectively, at day 22. The TGD's were 29 and 26days at a tumor volume of 800 mm³ for ITE and ITK, respectively. Therewas no obvious net body weight loss (data not shown) even though therewas 1 out of 8 mice in ITK group died at day 32.

Both ITE and ITK at 80 mg/kg (i.p. once daily) again demonstrated asimilar efficacy in inhibiting human ovarian cancer (OVCAR-3) growth(FIG. 2C). The TGFs were 47% (p<0.002, n=8) and 46% (p<0.001, n=8) atday 33 for ITE and ITK, respectively. The TGD's for ITE and ITK were,respectively, 10 and 13 days at a tumor volume of 800 mm³. There was noobvious net body weight loss (data not shown). There was again 1 out of8 mice in ITK group died also at day 32.

ITE displayed a modest efficacy, albeit at a modest dose (20 mg/kg, i.p.once daily), in inhibiting human breast cancer (MCF-7) growth (FIG. 2D).A TGI was calculated as 71% (p<0.031, n=8) at day 19 for the ITEtreatment. A TGD of 3 days at a tumor volume of 500 mm³ was obtained forITE group. Further increase in dosing levels is certainly needed toyield a better growth inhibition and delay but the response of MCF-7xenografts to ITE treatment was there.

The performance of one of ITE structural analogs (ITK) in this Examplevalidates a huge potential of ITE analog development based on aframework specified by the Structural Formula 4. In addition, an analogwith a thiol ester functional group replacing the normal (oxygen) esterin the ITE structure is envisioned to be specially important given theresults of ITK (Structural Formula 2) studies. The thiol (S, sulfur)ester so specified by the Structural Formula 3 is thus abbreviated asITSE. The structural feature of both ITK and ITSE may help fend againstattacks by numerous esterases in biological systems specifically to theoxygen ester functional group on the structure of ITE.

Example 3 Materials and Methods

Murine Lewis lung cancer cell line LLC (ATCC) was cultured as describedfor LNCaP in Example 1 except DMEM, instead of RPMI-1640, medium beingused. Each female C57BL/6 mice, 6 to 8 weeks of age, was inoculated with3×10⁵ LLC cells in 0.1 ml of PBS for tumor development. ITE treatmentwas started when a mean tumor volume reached 80 to 120 mm³. All theother materials and methodologies were the same as that described inExample 1.

Results and Discussion

The benefit of using xenograft model is that human cancers can bedirectly tested on animals. The disadvantage, however, is that the micehave to have defects in their immune systems so that they will notreject human cancer cells. It is obvious, therefore, that this type ofmodels cannot be used to test if ITE can stimulate immune system todramatically enhance its therapy. A syngeneic model, mouse tumor cellsinoculated to mice with healthy immune systems, was then used. At a doseof 20 mg/kg b.w. (i.p. once daily), while ITE showed a growth inhibitionof the mouse lung cancer (n=8, TGI=65% at day 15, FIG. 3A), there was noindication of cancer elimination or eradication. In fact, the tumorgrowth was so aggressive in this model that the experiment had to bestopped earlier to relieve the suffering of animals in both control andITE groups from heavy tumor burdens.

At a dose of 80 mg/kg b.w. (i.p. once daily), ITE significantly improvedthe growth inhibition of tumors so that ITE treated group could then bekept to the end of the experiment without early termination (FIG. 3B)like before. The TGI at day 20 for ITE treatment was 42% (n=8, p<0.037)and TGD at a tumor volume of 1000 mm³ was 7 days. One of the mice in ITEgroup (mouse No. 33) started to shrink its tumor upon the start of ITEtreatment and kept doing so until its tumor was no longer palpable atday 13 (FIG. 3C). The mouse kept tumor free during the rest of the ITEtreatment phase (total 28 days). One more month was then given to themouse after the stop of the 28-day treatment to show possible regrowthof its tumor. But that did not happen and the mouse kept tumor freeduring the entire month of observation, suggesting the elimination ofevery cancer cell by the treatment (FIG. 3C). Body weight changemonitoring indicated the mouse No. 33 and the other mice in ITE grouptolerated well to the treatment (FIG. 3D).

With xenograft models, complete tumor elimination has never happened ata dose of 80 mg/kg b.w. (i.p. once daily) or even at 80 mg/kg dosed(i.p.) twice a day. That may argue for the stimulation of immune systemsin mice of this syngeneic model. Actually, results in Example 4 belowmay also support this notion by showing increased counts of white bloodcells, neutrophils, lymphocytes, and platelets at high (500 mg/kg, i.p.once daily) and mid (100 mg/kg, i.p. once daily) but not low (20 mg/kg,i.p. once daily) dose. Cancer eradication thus may be achieved if immunesystem could be mobilized to help fighting cancers and cleaning upindividual cancer cells while cancer growth could be effectivelyinhibited and assaulted at the same time.

Example 4 Material and Methods

ITE nano-suspension was prepared by milling ITE powder in watercontaining 1% CMC-Na (sodium carboxymethyl cellulose), 0.5% SLS (sodiumlauryl sulfate), 0.085% PVP K90 (polyvinylpyrrolidone K90), and 0.2%Benzoate with a Media Wet Milling Machine (Dispermat SL-nano, WAB WillyA. Bachofen A G, Muttenz, Switzerland) until reached a desired sizerange. The particle size was determined by a Laser Diffraction ParticleSize Analyzer (MS2000, Malvern Instruments, Worcestershire, UK). Theparameters of particle sizes were determined as D10 (diameter of 10% ofparticles)=67 nm, D50=114 nm, and D90=207 nm. The nano-suspension thusprepared was stored at 4° C. until use.

Female C57BL/6 mice, 6 to 8 week old, were randomly assigned to 4 dosegroups (0, 20, 100, and 500 mg/kg b.w.) each with 6 animals. ITEnano-suspension was administered by i.p. injection once daily for 7consecutive days. Mortality, clinical signs, body weight, and foodconsumption were recorded. Data on hematology (3 of 6 mice) and serumchemistry (the other 3 of 6 mice) were collected. TK (Toxicokinetic)parameters were determined as described in Example 1 and plasma levelsof ITE at both 1 and 3 hr. post dosing on day 1, 3, and 7 were measured.A gross observation of major organs at necropsy was conducted.

Results and Discussion

TK data confirmed the proper ITE system exposure (data not shown). Therewas no mortality observed except that one mouse in 20 mg/kg b.w. (lowdose) group died without known cause before the second day of dosing.There was no significant body weight loss due to ITE treatments eventhough a dramatic decrease in food consumption in all 3 ITE treatedgroups was noticed at day 1 of the study. No abnormality was observedfrom major organ inspection of all the ITE treated groups at necropsy.Levels of ALT (alanine aminotransferase), AST (aspartateaminotransferase), and TP (total protein) of 500 mg/kg (high dose) groupwas raised by 3.2 (p<0.05), 1.8 (not significant), and 1.2 (p<0.05)folds, respectively, over vehicle control (Table 1). BUN (blood ureanitrogen) of 20 mg/kg (low dose) group was raised by 1.4 folds (p<0.05)over vehicle. These data, especially that of ALT, may suggest anapproaching near to the up limit of ITE dosing. WBC (white blood cellcount) was raised by 2.6 (p<0.05) and 2.0 folds (not significant) for100 (mid) and 500 mg/kg (high) group, respectively. Others likepercentage of PLT (platelets), percentage of NEUT (neutrophils), numbersof neutrophils (#NEUT), and numbers of lymphocytes (#LYMPH) wereincreased in both 100 and 500 mg/kg groups albeit not statisticallysignificant (Table 1). Data in hematology, even though more confirmatorystudies need to be done, may actually suggest the mobilization of theimmune system by ITE, thus reverberating probably to the data of cancereradication presented in Example 3.

TABLE 1 Partial readings on hematology and serum chemistry Dose ALT ASTBUN TP WBC PLT NEUT #NEUT #LYMPH (mg/kg) (U/L) (U/L) (mmol/L) (g/L)(×10⁹ cells) (×10⁹ cells) (%) (×10⁹ cells) (×10⁹ cells) 0 23.6 (6.4) 84.3 (37.9) 7.6 (1.2) 39.7 (3.3) 4.22 (1.85) 539 (218) 19.4 (4)   0.87(0.67) 3.09 (1.82) 20  43.2 (43.7) 118.1 (41.7) 10.5* (0.3)  39.5 (2.7)4.68 (0.04) 435 (295) 23.3 (no) 1.08 (no) 3.35 (no)  100  33.2 (10.2)131.6 (27.8) 7.3 (1.6) 40.8 (3.3) 10.77* (4.26)  668 (202) 46.6 (no)3.62 (no) 3.5 (no)  500 74.5* (21.6) 151.2 (37.1) 9.1 (0.3) 49.5* (1.5) 8.38 (1.32) 1075 (99)   40.5 (3.4)   3.4 (0.64) 4.81 (0.81) Table 1displays partial readings on hematology and serum chemistry, wherein thelisted are group means with SD (standard deviation) inside theparentheses, and wherein the * depicts a statistical significance (p <0.05), and wherein the “no” means a standar d deviation is not availabledue to sample size, and wherein ALT means alanine aminotransferase, ASTasparta te aminotransferase, TP total protein, BUN blood urea nitrogen,WBC white blood cell count, PLT platelet, NEUT neutrophils, #NEUT numberof neutrophils, an d #LYMPH number of lymphocytes.

INDUSTRIAL APPLICABILITY

The present invention can be applied to the area of cancer interventionor eradication for human beings and other animals, especially mammals.

REFERENCE SIGNS LIST

As used herein, the term “structural analog” or simply “analog” of ITEis defined as a compound with chemical structure similar to that of AhRendogenous ligand ITE.

As used herein, the term “alkyl” represents a group of hydrogensaturated one to six carbons connected in either straight or branchedfashion.

As used herein, the term “haloalkyl” represents an alkyl substituted byone or more halogen atoms.

As used herein, the term “alkenyl” represents a group of hydrocarbonscontaining two to six carbons connected in either straight or branchedfashion with at least one carbon-to-carbon double bond.

As used herein, the term “alkynyl” represents a group of hydrocarbonscontaining two to six carbons connected in either straight or branchedfashion with at least one carbon-to-carbon triple bond.

As used herein, the term “halo” represents any of halogen atoms (F, Cl,Br, or I).

As used herein, the term “carbonyl” represents:

As used herein, the term “alkanoyl” represents an alkyl connected to acarbonyl group:

As used herein, the term “haloalkanoyl” represents a haloalkyl connectedto a carbonyl group:

As used herein, the term “nitrogen protective group” represents groupscommonly used to protect nitrogen from undesired chemical reactionsduring synthesis procedures.

As used herein, the term “amino” represents NR_(a)R_(b) where R_(a) andR_(b) can be independently selected from hydrogen, halo, formyl (—CHO),alkyl, haloalkyl, alkenyl, alkynyl, alkanoyl, haloalkanoyl, or anitrogen protective group.

As used herein, the term “alkoxy” represents an alkyl connected to anoxygen atom (—O-alkyl).

As used herein, the term “haloalkoxy” represents a haloalkyl connectedto an oxygen atom (—O-haloalkyl).

As used herein, the term “thioalkoxy” represents an alkyl connected to asulfur atom (—S-alkyl).

As used herein, the term “carbonyloxy” represents an alkanoyl connectedto an oxygen atom:

CITATION LIST Patent Literature

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1-12. (canceled)
 13. A method of treating cancer in a patient,comprising administering a therapeutically effective amount of2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE)or a structural analog thereof to the patient, wherein ITE hasstructural formula 1:


14. The method of claim 13, wherein the ITE structural analog is acompound of structural formula 2:


15. The method of claim 13, wherein the ITE structural analog is acompound of structural formula 3:


16. The method of claim 13, wherein the ITE structural analog is acompound of structural formula 4:

wherein: X and Y are independently selected from the group consisting ofO (oxygen) and S (sulfur); R_(N) is selected from the group consistingof hydrogen, halo, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,alkanoyl, haloalkanoyl, and a nitrogen protective group; R₁, R₂, R₃, R₄,and R₅ are independently selected from the group consisting of hydrogen,halo, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,amino, nitro, alkoxy, haloalkoxy, thioalkoxy, alkanoyl, haloalkanoyl,and carbonyloxy; R₇ is selected from the group consisting of hydrogen,halo, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,amino, nitro, alkoxy, haloalkoxy, and thioalkoxy; and R₆ is

wherein R₈ is selected from the group consisting of hydrogen, halo,cyano, alkyl, haloalkyl, alkenyl, and alkynyl; or R₆ is

wherein R₉ is selected from the group consisting of hydrogen, halo,alkyl, haloalkyl, alkenyl, and alkynyl; or R₆ is

wherein R₁₀ is selected from the group consisting of hydrogen, halo,hydroxy, thiol, cyano, alkyl, haloalkyl, alkenyl, alkynyl, and nitro; orR₆ is

wherein R₁₁ is selected from the group consisting of hydrogen, halo,alkyl, haloalkyl, alkenyl, and alkynyl.
 17. The method of claim 13,further comprising administering to the patient another cancertherapeutic agent.
 18. The method of claim 13, further comprisingadministering maintenance doses of ITE or the ITE structural analogwhile the patient is in remission.
 19. A method of stimulating theimmune system in a patient in need thereof, comprising administering atherapeutically effective amount of ITE or a structural analog thereofto the patient, wherein ITE has structural formula 1:


20. The method of claim 19, wherein the ITE structural analog is acompound of structural formula 2:


21. The method of claim 19, wherein the ITE structural analog is acompound of structural formula 3:


22. The method of claim 19, wherein the ITE structural analog is acompound of structural formula 4:

wherein: X and Y are independently selected from the group consisting ofO (oxygen) and S (sulfur); R_(N) is selected from the group consistingof hydrogen, halo, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,alkanoyl, haloalkanoyl, and a nitrogen protective group; R₁, R₂, R₃, R₄,and R₅ are independently selected from the group consisting of hydrogen,halo, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,amino, nitro, alkoxy, haloalkoxy, thioalkoxy, alkanoyl, haloalkanoyl,and carbonyloxy; R₇ is selected from the group consisting of hydrogen,halo, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,amino, nitro, alkoxy, haloalkoxy, and thioalkoxy; and R₆ is

wherein R₈ is selected from the group consisting of hydrogen, halo,cyano, alkyl, haloalkyl, alkenyl, and alkynyl; or R₆ is

wherein R₉ is selected from the group consisting of hydrogen, halo,alkyl, haloalkyl, alkenyl, and alkynyl; or R₆ is

wherein R₁₀ is selected from the group consisting of hydrogen, halo,hydroxy, thiol, cyano, alkyl, haloalkyl, alkenyl, alkynyl, and nitro; orR₆ is

wherein R₁₁ is selected from the group consisting of hydrogen, halo,alkyl, haloalkyl, alkenyl, and alkynyl.
 23. The method of claim 19,wherein the patient has an increased count of cells selected from thegroup consisting of white blood cells, neutrophils, lymphocytes, andplatelets after the administering step.
 24. A compound of structuralformula 3:


25. A pharmaceutical composition comprising the compound of claim 24 anda pharmaceutically acceptable carrier.
 26. A compound of structuralformula 4:

wherein: X and Y are independently selected from the group consisting ofO (oxygen) and S (sulfur); R_(N) is selected from the group consistingof hydrogen, halo, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,alkanoyl, haloalkanoyl, and a nitrogen protective group; R₁, R₂, R₃, R₄,and R₅ are independently selected from the group consisting of hydrogen,halo, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,amino, nitro, alkoxy, haloalkoxy, thioalkoxy, alkanoyl, haloalkanoyl,and carbonyloxy; R₇ is selected from the group consisting of hydrogen,halo, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, alkynyl,amino, nitro, alkoxy, haloalkoxy, and thioalkoxy; and R₆ is

wherein R₈ is selected from the group consisting of hydrogen, halo,cyano, alkyl, haloalkyl, alkenyl, and alkynyl; or R₆ is

wherein R₉ is selected from the group consisting of hydrogen, halo,alkyl, haloalkyl, alkenyl, and alkynyl; or R₆ is

wherein R₁₀ is selected from the group consisting of hydrogen, halo,hydroxy, thiol, cyano, alkyl, haloalkyl, alkenyl, alkynyl, and nitro; orR₆ is

wherein R₁₁ is selected from the group consisting of hydrogen, halo,alkyl, haloalkyl, alkenyl, and alkynyl.
 27. The compound of claim 26,wherein X is S (sulfur).
 28. The compound of claim 26, wherein R_(N) iscyano.
 29. A pharmaceutical composition comprising the compound of claim26 and a pharmaceutically acceptable carrier.
 30. A pharmaceuticalcomposition comprising the compound of claim 27 and a pharmaceuticallyacceptable carrier.
 31. A pharmaceutical composition comprising thecompound of claim 28 and a pharmaceutically acceptable carrier.